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Int..I. Dev. Billi. 33: 389-395 (1989)
Origillal
Arlirle
Immunohistochemical analysis
process
of the quail
MALKA GINSBURG',
Department
of Zoology,
JACOB
Institute
of the segregation
germ cell lineage
HOCHMAN
of Life Sciences.
and HEFZIBAH EYAL-GILADI
The Hebrew
University
of Jerusalem,
Israel.
ABSTRACT
An antiserum
against
quail 7 day gonadal
germ cells was found to react
specifically
with gonadal germ cells of both sexes. Transverse
sections from a range of early
quail developmental
stages were submitted
to the antibody
PAP reaction.
Blastodiscs
from
the earliest uterine stages (II to X E.G & K) reacted very strongly, while the overall reaction
gradually
decreased
in older blastoderms.
At stage XIII both epiblast
and hypoblast
were
weakly stained,
but some large, PGC-like cells stained intensively.
During gastrulation
(PS
formation)
the reaction
of the epiblast
disppears
quicker than that of the hypoblast.
The
newly formed
mesoderm
and entoderm
do not react at all and the reaction
gradually
becomes
limited mainly to the PGCs and somewhat
to the primary
hypoblast
which is
moving into the germinal
crescent.
The widely spread reaction
at the early stages is thus
gradually
being restricted
to the PGCs.
KEY WORDS:
germ rl'll.~, avian
gl'nJl line, imlllllllOhistochemistJ)
Introduction
Among the vertebrates,
only in the anuran embryo
has there been detected
a germ plasm which is thought
to contain
the germ cell lineage
determinants
(Bounoure, 1934; Whitington
and Dixon, 1975). No distinct
germ plasm has ever been detected
either in the zygote
or in specific blastomeres
of the other groups, and the
mode and time of primordial
germ cell (PGC) determination are still an enigma.
Studies on the origin of the PGCs and the germ cell
lineage
in avian embryos
have been limited
by the
morphological
homogeneity
of early avian cell populations. The histochemical
PAS staining for glycogen
has
so far been the only useful marker for differential
labeling of the PGCs in early chick embryos (Meyer. 1959;
Fujimoto
et al., 1976; Ginsburg
and Eyal-Giladi.
1986,
1987). However,
even this marker allows identification
only at primitive
streak (PS) stages and onwards.
Indirect experiments
have demonstrated
the epiblastic
origin of the PGCs IEyal-Giladi
et al., 19811 and the existence of at least some determined
PGCs already
at
stages XI-XII E.G&K (Sutasurja
et al., 1983; Ginsburg
and Eyal-Giladi,
1986, 1987).
Recently,
two monoclonal
antibodies
were found
which recognize
avian PGCs: the first one, QH1, was
raised against 12-day quail bone marrow.
It was found
"Address
for reprints:
0214-6282/89
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Printcd in Sp~in
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Department
of Zoology,
Institute
of Life Sciences.
to bind to cells of the hemangioblastic
lineage and also
to recognize
PGCs of the young
quail blastoderm
IPardanaud
et al., 19871. A second
antibody,
EMA-1.
which was raised originally
against
mouse embryonal
carcinoma
cells (Hahnel and Eddy, 1982, 19861, also recognized
chick PGCs from the time of their detachment
from the epiblast,
and during their migration
and colonization of the gonadal
germinal
epithelium
(Urven et
al., 19881.
We have taken a direct approach
to address the question of PGC lineage using antibodies
that were raised
against quail gonadal PGCs. These were used to analyze
histologic
sections
of consecutive
stages of quail embryos from early cleavage
(uterine stages) to 7 days of
incubation.
Results
Since the PGCs for immunization
were obtained
from
day 7 quail embryonic
gonads,
we first studied
the
interaction
of the immune
serum
(from consecutive
bleedings
- see Table 1) with embryonic
tissues
of the
same stage. Positive reactions
with the immune serum
were consequently
obtained
from the fourth bleeding
(841 and on 185,861. The rabbit was sacrificed after 86,
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Fig. 1. Paraffin sections of 7 day quail embryonic gonads. x 480. (A) PAP reaction of 84 1.-100 on a section of female gonad. (B) PAP secrion of 84
1:100 on a section of male gonad IC) PAP reacrion of rhe preimmune 80 1:100 on a section of male gonad. (D) A section of male gonad that was
hematoxylin
stained
following
the immunohistochemical
reaction.
Segregatio/1 of quail germ cdls
Fig. 2. Paraffin sections
female gonad.
but throughout
84 was used.
Gonadal
of 15 day quail embryonic
the present
study
gonads.
only immune
39l
x 480. !A) PAP reaction of 84 1:100 on male gonad (81 PAP reaction of 84 1:100 on
serum
PGCs
Immune
serum
at dilutions
of 1:100-1:500
reacted
specifically
with the cytoplasm
of the undifferentiated
germ cells of both sexes which populated
the 7 day
embryonic
gonads.
No reaction
was detected
in the
mesodermal
cells constituting
the gonadal tissues (Fig.
1A. 8, CI. Other tissues
taken from 7 day old quail
embryos
were also sectioned
and tested with different
dilutions
of 84 antiserum.
Limbs, heart and liver were
negative.
Yolk sac endoderm
was the only tissue that
reacted with the antiserum
at dilutions
of 1:100-1:500.
The preimmune
serum
(80) was inactive
against
all
target
tissues
tested.
Both preimmune
and immune
sera reacted with capillary endothelial
cells (Fig. 1). As
both frozen and paraffin sections demonstrated
comparable results we restricted
all subsequent
tests to paraffin sections,
which were histologically
much better preserved.
Quail gonadal
PGCs were immunohistochemically
reactive until at least 15 days of incubation,
when the reaction was more prominent
for male than for female
PGCs (Fig. 2A. 81. Older gonads were not checked. To
check for species
specificity,
chick embryonic
gonads
were tested with the antiserum
to quail PGCs. No reaction was detected
in sections
of 10 day old chick embryos, a stage comparable
to 7 day old quail embryos.
Uterine
stages
{II, VII, X E.G&K}
All the cells in sections
of these stages reacted very
strongly
and invariably
with B4 antiserum
at a dilution
of 1:2000 (Fig. 3A). Preimmune
serum at the same dilution gave entirely negative
results (Fig. 38).
Stage
XIII
{blastula}
The 84 immune serum reacted equally with both the
epiblast
and the hypoblast
at dilutions
up to 1:30,000.
After absorption
on liver cells from 7 day quail embryos
(1 h, 4CC) the B4 immune
serum was applied
to the
sections
at a dilution
of 1:1000. The absorbed
serum
distinguished
some large, germ cell-like
cells in the
hypoblast
that stained more intensely
than their neighbors (Fig. 3CI.
Gastrulating
stages
During gastrulation
the epiblast
lose its immunostainability
with
seemed to gradually
the 84 antiserum.
In
392
At. GillslJllrg et al.
A
B
~
-
<"'T...!:II
""r..,~
Fig. 3. Paraffin sections of quail at various developmental
stages. x 300.(AJ PAP reaction of 84 antiserum 1:2000 on section
of the preimmune 80 antiserum 1:2000 on section of stage VII blastoderm. IC) PAP reaction of 84 antiserum
cells, at dilution of 1:1000. on transverse section of stage XIII blastoderm, followed by hematoxylin staining. (DI PAP reaction
on transverse section made at the area of Hensen's node of stage 3 (H&H) blastoderm, followed by hematoxylin staining.
IB) PAP reaction
antiserum 1:500 at the area of the PS, postenor to D. IF) The same section as
the vicinity of the PS (Fig. 3E) several cells were detected in the epiblast
which expressed
a weak reaction
mainly
in their basal parts. The newly formed
mesoderm did not react with the antiserum
except for a few
scattered cells situated lateral to the PS (Fig. 3F). Also,
the definitive
endoderm
(EN) underneath
the PS and
lateral to it reacted
negatively
with the antiserum.
In
Hensen's
node (HN) at the anterior
end of the PS the
reaction is very weak (Fig. 3D) and it entirely disappears
from the epiblastic
area anterior to the streak. However,
underneath
the non-reactive
HN and more anterior to it,
the lower layer stands out due to its positive
reaction
In
of stage VII blastoderm
after absorption on liver
of 84 antiserum 1:500
(EI PAP reaction of 84
E photographed lateral to the PS
with 8411:5001
antiserum
(Fig. 3D). Similarly,
several
PGCs with an even more prominent positive reaction
can be distinguished in the space between the epiblast
and the hypoblast.
Discussion
The timing as well as the mechanism
of the segregation of the germ line from the somatic cell Jines in vertebrates
is still unknown.
With the exception
of the
anurans
(Czolowska,
1972; Bounoure,
1934; Blackler,
1958, 1966; Whitington
and Dixon, 1975; Ressom
and
Segregation
TABLE 1
SCHEDULE
Days
1
14
20
36
56
76
96
OF RABBIT BLEEDINGS AND IMMUNIZATIONS
WITH PGCs
Number ot PGCs injected
16
8.4
21.5
20
27
20
x 1O~
X 104
X 104
X 104
X 104
X 104
Bleeding
sample
Bo
B1
B2
B3
B4
B5
B6
Dixon, 1988), in oocytes or cleaving embryos of all
other vertebrates
no identifiable
germ plasm was ever
detected.
Moreover
it has been shown that in contrast
to anurans, the PGCs of Urodeles (Sutasurja
and Nieuwkoop, 1974), birds (Eyal-Giladi
et a/., 19811 and mammals (Jacob, 1977; Mintz, 1971; Snow and Monk, 1983)
are of epiblastic
origin.
As to the mode of determination
of the PGCs in the
ectodermal-epiblastic
layer, Sutasurja
and Nieuwkoop
(1974) claimed that in Urodeles the PGCs are induced in
the ectodermal
part of the animal hemisphere,
by the
ventral half of the vegetal
hemisphere,
implying
that
the animal cap is totipotent
and capable
of forming
PGCs if exposed
to the appropriate
induction.
In mammals
there is still a discussion
as to whether
the epiblast is composed
of a totipotent
cell population
in which the somatic
cell population
is the one first
determined
by yet unknown
restrictive
events.
This
would leave a small population
of unrestricted
totipotent cells to become
PGCs later on (McMahon
et al.,
1983). Another approach
favors the idea (Soriano
and
Jaenisch,
1986; Monk et a/., 19871 that it is the germ line
which is first allocated.
In birds, Ginsburg
and EyalGiladi (1986,19871
have demonstrated
that most PGCs
segregate
from the central
part of the epiblast.
This
leaves the question
open whether the epiblast contains
specific
determinants
that were allocated
to it by a
specific
cytoplasmic
section
of the germinal
disc, or
whether the central area of the epiblast is exposed to inductive influences
dictating
the formation
of the PGCs.
of quail germ cells
393
the germ line at earlier
stages
remained
unclear.
A
broader view regarding
the early stages evolved from
studies
using a monoclonal
antibody
which was produced gainst a Forssman-like
antigen
present
on the
membranes
of teratocarcinoma
stem cells as well as on
PGCs (Willison and Stern, 1978; Evans eta/., 1979). This
antibody
reacted
positively
with the entire inner cell
mass in the early developmental
stages.
However,
in
somewhat
later stages
it stopped
reacting
with the
ectoderm
and was restricted
only to the extraembryonic endoderm
and the gonadal
PGCs.
In Avians, the monoclonal
antibody
QH1 (Pardanaud
et al., 1987) shows
an affinity to quail blastomeres,
ranging between
stages X E.G&K to 13 H&H, identified
by the authors as PGCs. Unfortunately,
it was not mentioned whether
the above antibody,
which is not specific to the germ line, was tested on earlier embryos.
The
same is true of the studies
using the monoclonal
antibody EMA-1 (Hahnel and Eddy, 1982, 1986) on chick
cells (Urven et al., 1988). This antibody
reacted
with
some of the epiblastic
cells at stages X-XI E.G&K and
with some ingressing
cells at stage XII. However,
the
identity
of the reactive
cells as PGCs could not be
established
at such early sages,
due to the lack of
morphological
criteria. Only at stage XIV and onwards,
could the positive EMA-1 cells be shown to be differentially PAS positive. The chick PGCs remained
reactive to
EMA-1 throughout
the migration
period and the colonization of the gonad. This study seems to support
the
notion of the epiblastic
origin of the chick PGCs but
does not contribute
information
as to their earlier origin. On the other hand, it demonstrates
for the first time
the existence
of common
antigens
shared by the germ
lineage of chicken and mouse.
Several investigators
have tried to tackle the dilemma of PGC origin and determination
by tracing the developmental
pathway of the germ cell line, using an immunological
approach.
Most of the studies mentioned
above used polyclonal
or monoclonal
antisera
raised against
cells other than
the germ line. Our approach,
like that of Heath (19781,
was to use an anti-PGC serum in order to try and detect
a possible
unique cytoplasmic
antigen
hopefully
restricted to a specific region in the quail's germ which will
prove to be connected
to the germ line. What we found,
however,
was exactly the opposite,
namely,
a widespread
reactivity
of the early germ to the antiserum,
which gradually
became restricted
to PGCs on the one
hand, and to the primary hypoblast
of the other. This
result closely resembles
the results with the Forssmanlike antigen in the mouse, which reacts with the entire
ICM of a 3'/ -4'/, day embryo, while later on the epiblast
loses its r~activity,
which remains
strongly
positive
only in the endoderm
and the PGCs.
In the mouse, polyclonal
antibodies
were produced
either against stem cells of teratocarcinoma
(Artzt etal.,
1973; Gachelin et a/., 19761 or against germ cells (Heath,
1978). The results suggest
that the germ cell lineage is
derived
directly from the embryonic
ectoderm
of the
early implantation
mouse embryo. However, the fate of
It is worthwhile
mentioning
in this connection
that
the primary
hypoblast
of the quail's blastula
(stage X
E.G&K), which in our case reacted positively,
continued
to be positive
also during the hypoblast's
anteriorly
directed
movement
onto the germinal
crescent
and the
yolk sac.
--
394
M. Ginshurg et a!.
Considering
all the above information
it seems that
the different
antigens
which were shown to be connected to PGCs can be classified
into two groups.
One
group of antigens
was detected
at post cleavage
stages
iPardanaud
et al., 1987; Urven et al., 19881 and their
appearance
seems to be correlated
to already morphologically
recognizable
PGC differentiation.
Some of
these antigens
may even be connected
with cell migration or ingression.
Other antigens,
like the Forsmannlike antigen and the ones which react with our polyclonal antiserum,
seem to be "pluripotent
antigens"
present at early cleavage.
much before the appearance
of
identifiable
PGCs. These antigens
are initially uniformly
dispersed.
They gradually
disappear
from all the cells
commited to become somatic, while they are retained in
the PGCs and the primary hypoblast, which in birds is
believed to be involved in the translocation
of the PGCs
into the germinal
crescent
(Ginsburg
and Eyal.Giladi,
1986).
The results of the present study cannot be regarded
as conclusive.
However,
since we used a polyclonal
antoiserum
raised
against
whole
gonadal
PGCs we
stand a better chance of seeing our antiserum
react with
more than one antigen specific for PGCs.
All the evidence
concerning
the early stages both in
mouse and quail indicates
that during cleavage
there is
still no predetermined
germ plasm and that all the pluripotent cells are potential
PGCs. This agrees also with
the conclusions
of Sutasurja
and Nieuwkoop
(1974)
concerning
the Urodeles.
Our present data cannot distinguish
between the two
approaches
mentioned
above: 1) that the somatic cells
are determined
first, leaving a stock of pluripotent
cells
as PGCs; 2) that some active inductive
influence
is
needed to preserve
the pluripotency
of the PGCs, which
would otherwise
be restricted
to becoming
somatic
cells.
Materials
and
Methods
Immunization
For immunization,
quail gonadal
PGCs from embryos
of 7
days of incubation
were used. The isolated
gonads
of both
sexes
were cleaned
of adhering
tissues
and then gently
pressed
between
the bottom of a Petri dish and a coverslip
(Heath,
1978) to the point of rupture.
The cell suspension
containing
the fluid that was extruded
from the gonads was
filtered through
a double layer of gauze and centrifuged
for 10
min at 800xg. 90% of the pelleted
cells were PGCs according
to morphological
criteria. The cells were immersed
for 10 min
in 1% formaldehyde,
washed
in PBS and injected
intradermally in complete
Freund's
adjuvant
into multiple sites of an
adult female rabbit.
The protocol
Table 1.
for immunization
and the bleeding
is shown
in
Preimmmune
as well as immune
sera
56~C for 30 min and stored at -20~C.
Immuno-peroxidase
staining
were
inactivated
at
on sections
Frozen sections
Blastoderms
and tissues were fixed for 10 min in 3% paraformaldehyde
in PBS and washed
in PBS. They were then
incubated
for 30 min in 0.1% glycine in PBS, whashed
in PBS
and then frozen in liquid nitrogen
vapors. 6 11m-thick sections
were cut by a kryostat
and fixed on the slides for 5 min in
acetone
at _20°.
Paraffin
sections
Blastoderms
and tissues were fixed for 1 h in Brodski's fluid
(1960) and then embedded
in paraffin.
6 11m-thick sections
were put on slides previously
coated with gelatine.
Cellular
antigens
were visualized
using the peroxidase
anti-peroxidase
technique,
essentially
described
by Sternberger (1979). Some slides were also stained with hematoxylin following
the immunohistochemical
reaction.
All the chemicals were purchased
from Miles Veda, Rehovot,
Israel.
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